The binding properties of a protein largely depend on the exposed surface amino acid residues of its polypeptide chain (see, for example, Bruce Alberts et al., “Molecular Biology of the Cell”, 2nd edition, Garland Publishing, Inc., New York, 1989; and H. Lodish et al., “Molecular Cell Biology”, 4th edition, W. H. Freeman and Company, 2000). These amino acid residues can form weak noncovalent bonds with ions and molecules. Effective binding generally requires the formation of many weak bonds at a “binding site,” which is usually a cavity in the protein formed by a specific arrangement of amino acids. There must be a precise fit with the binding site for effective binding to occur.
Pharmaceutical chemicals are the active ingredients in drugs, and it is believed that their therapeutic properties are linked to their ability to bind to one or more binding sites. The shapes of these binding sites may differ greatly among different proteins, and even among different conformations of the same protein. Even slightly different conformations of the same protein may differ greatly in their binding abilities. For these reasons, it is extremely difficult to predict which chemicals will bind effectively to proteins. Research and development for a new pharmaceutical chemical for a drug, i.e. drug development, generally involves determining the binding affinities between a potential pharmaceutical chemical (preferably a water soluble organic chemical that can dissolve into the blood stream) and a target binder (generally a biological material such as an enzyme or non-enzyme protein, DNA, RNA, human cell, plant cell, animal cell, and the like) at many stages of the drug development process. The target binder may also be a microorganism (e.g. prion, virus, bacterium, spores, and the like) in whole or in part. The drug development process typically involves procedures for combining potential pharmaceutical chemicals with target binders, detecting chemical binding between the potential pharmaceutical chemicals and the target binders and determining the binding affinity and kinetics of binding of a target binder to a chemical to form a complex or the kinetics of release of a bound chemical from a complex. The binding affinity is defined herein as the associative equilibrium constant Ka, where Ka is defined by equation (1) below.Ka=[complex]/[target binder][potential pharmaceutical chemical]  (1)In equation (1), [complex] is the concentration in moles per liter of the target binder/potential pharmaceutical complex, [target binder] is the concentration in moles per liter of the target binder, and [potential pharmaceutical chemical] is the concentration in moles per liter of the potential pharmaceutical chemical. Nowadays, the drug development process may involve the rapid screening of hundreds or thousands of potential pharmaceutical chemicals in order to identify a “lead compound,” which is one of the many tested that binds very strongly, i.e. has a high binding affinity, with a particular target binder. After such a lead compound has been identified, then other potential pharmaceutical chemicals similar in structure to the lead compound are synthesized and tested in order to determine which of these potential pharmaceutical chemicals, if any, exhibits an even higher binding affinity. Some screening methods are described in the following patents, all of which are hereby incorporated by reference.
U.S. Pat. No. 6,147,344 to D. Allen Annis et al. entitled “Method for Identifying Compounds in a Chemical Mixture”, which issued Nov. 14, 2000, describes a method for automatically analyzing mass spectrographic data from mixtures of chemical compounds.
U.S. Pat. No. 6,344,334 to Jonathan A. Ellman et al. entitled “Pharmacophore Recombination for the Identification of Small Molecule Drug Lead Compounds,” which issued Feb. 5, 2002, describes a method for identifying a drug lead compound by contacting target biological molecules with cross-linked binding fragments.
U.S. Pat. No. 6,395,169 to Ole Hindsgaul et al. entitled “Apparatus for Screening Compound Libraries,” which issued May 28, 2002, describes an apparatus that employs frontal chromatography combined with mass spectrometry to identify and rank members of a library that bind to a target receptor.
Some screening methods (fluorescence activated cell sorting, for example) cannot detect binding between many types of potential pharmaceutical chemicals (a non fluorescent antibody, for example) and a target binder unless the potential pharmaceutical chemical is provided with a chemical tag. Tagging may involve modifying the original chemical by attaching a “tag” (a chemical group that fluoresces when exposed to ultraviolet or visible light, for example) to a portion of the potential pharmaceutical chemical. Afterwards, the tagged chemicals are exposed to cells (muscle cells, for example) for a long enough period of time for binding to occur (if it does occur) to some of the cells to produce a cell/antibody complex. Afterwards, the cells are sent through a laser beam one at a time. The laser producing the beam is interfaced to a detector, such as an ultraviolet/visible fluorescence detector. The cell/antibody complexes produce a detectable fluorescence signal when exposed to the laser beam, and as the screening method proceeds, when a fluorescence signal is detected, the bound complexes that produce the signal are collected (see, for example, Bruce Alberts et al., “Molecular Biology of the Cell”, 2nd edition, Garland Publishing, Inc., New York, 1989, pages 159-160).
Another form of tagging involves attachment of the target binder to a surface. This type of tagging is used with surface plasmon resonance techniques (see, for example, U.S. Pat. No. 5,641,640 to A. Hanning entitled “Method of Assaying for an Analyte Using Surface Plasmon Resonance,” which issued Jun. 24, 1997; and U.S. Pat. No. 5,595,456 to M. Malmqvist et al entitled “Analyte Detection,” which issued Oct. 12, 1999, both hereby incorporated by reference).
It is generally assumed that the attachment of a fluorescent tag only serves to make visible to the instrument the otherwise invisible chemical and/or target binder, and that binding properties of the tagged and untagged materials are exactly the same. These assumptions may not be valid, as it is well known that even small changes to the structure of a chemical or target binder may affect its function. Tagged materials are structurally different from their untagged counterparts, and these structural differences could affect their binding properties.
An efficient method for screening chemicals (with potential pharmaceutical activity, for example) for binding to target binders remains highly desirable.
Therefore, an object of the present invention is to provide an efficient method for screening chemicals for binding to target binders.
Another object of the present invention is to provide a screening method that does not require modification of a potential pharmaceutical chemical with a chemical tag.
Additional objects, advantages and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following or may be learned by practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.